Despite numerous major scientific breakthroughs and tremendous R&D investments in the pharmaceutical sector over the past 3 decades, cancer still is the leading cause of disease-related mortality and awaits further advances toward improved therapy. Intensive effort has been focusing on the tumor suppressor p53 pathway because nearly all cancers show defects in this pathway, over 50% of which have mutations in the TP53 gene itself. We recently discovered Inauhzin (INZ) as a novel non-genotoxic p53 activator. INZ activates p53 by targeting SIRT1 and consequently suppresses tumor growth. In our ongoing studies to explore other possible targets of this compound, we identified a 78-kDa glucose-regulated protein (GRP78, also called BiP) as one of the top INZ target candidates. GRP78 is traditionally regarded as a major endoplasmic reticulum (ER) chaperone and a master regulator of the unfolded protein response (UPR) facilitating protein folding and assembly, protein quality control and regulating ER stress (ERS) signaling. Recent advances using mouse models and cellular approaches have revealed that GRP78 also functions beyond the ER. This discovery is not only critical for better understanding the unique and essential role of GRP78 in cancer development and prevention, but also offers an opportunity for cancer-specific targeting. Interestingly and surprisingly, we have also found that INZ can trigger the nuclear translocation of GRP78 that co-localized with accumulated p53 in the nucleus of cancer cells. Our finding suggests that INZ may regulate GRP78 through mechanisms distinct from the ERS-UPR pathway. Hence, the objectives of this application are to validate GRP78 as another bona fide target of INZ and to elucidate the mechanism underlying the role of GRP78 in INZ-induced p53 activation and anticancer activities. Our central hypothesis is that GRP78 is the direct tumor-specific target for INZ, leading to ER-independent activation of the p53 signaling pathway in cancer, but not normal, cells, and the combination of clinically used ERS/UPR-associated modulators with the p53 activator INZ may be a successful approach for overcoming chemoresistance and eliminating tumors. We will test this hypothesis by addressing the following Specific Aims: (1) to biochemically and biophysically characterize the specific binding of INZ to GRP78 in vitro and in cells; (2) to determine the role of cell-surface GRP78 in INZ uptake and specific cancer targeting, to determine how GRP78 is imported from ER to the nucleus by INZ to mediate p53 activation, and to evaluate the synergistic effect of INZ with clinically used ERS/UPR-associated modulators. Should our exploratory studies demonstrate GRP78 as a cancer specific target for INZ through an ER-independent p53 activation, this would not only provide proof-of-concept evidence for targeting both of the ER Stress/UPR related and p53 signaling pathways for cancer therapy, but would also offer new insights into the role of GRP78 in cancer development and therapy and have a strong impact on the area of molecule-specific anti- cancer drug discovery, particularly in the field of translational cancer research. 1